Adeno-associated virus virions with variant capsid and methods of use thereof

ABSTRACT

The present disclosure provides adeno-associated virus (AAV) virions with altered capsid protein, where the AAV virions exhibit greater infectivity of retinal cells, when administered via intravitreal injection, compared to wild-type AAV. The present disclosure further provides methods of delivering a gene product to a retinal cell in an individual, and methods of treating ocular disease.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.14/701,063, filed Apr. 30, 2015, which is a continuation of U.S. patentapplication Ser. No. 14/444,375, filed Jul. 28, 2014, which is acontinuation of U.S. patent application Ser. No. 14/113,205, filed Jan.22, 2014, which is a national stage filing under 35 U.S.C. §371 ofPCT/US2012/034413, filed Apr. 20, 2012, which claims the benefit of U.S.Provisional Patent Application No. 61/478,355, filed Apr. 22, 2011, eachof which applications is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.EY016994-02 and EY018241 awarded by the National Eye Institute of theNational Institutes of Health. The government has certain rights in theinvention.

BACKGROUND

Photoreceptors are the first neurons in the retina to receive andprocess visual information, converting visible electromagnetic radiationinto hyperpolarized responses through phototransduction. Theoverwhelming majority of inherited retinal diseases result in the lossof these cells, either directly, such as in dominant mutations thataffect rhodopsin protein folding, or indirectly, such as in recessivemutations that affect retinal recycling pathways in the retinal pigmentepithelium (RPE).

AAV belongs to the Parvoviridae family and Dependovirus genus, whosemembers require co-infection with a helper virus such as adenovirus topromote replication, and AAV establishes a latent infection in theabsence of a helper. Virions are composed of a 25 nm icosahedral capsidencompassing a 4.9 kb single-stranded DNA genome with two open readingframes: rep and cap. The non-structural rep gene encodes four regulatoryproteins essential for viral replication, whereas cap encodes threestructural proteins (VPI-3) that assemble into a 60-mer capsid shell.This viral capsid mediates the ability of AAV vectors to overcome manyof the biological barriers of viral transduction-including cell surfacereceptor binding, endocytosis, intracellular trafficking, andunpackaging in the nucleus.

LITERATURE

U.S. Patent Publication No. 2005/0053922; U.S. Patent Publication No.2009/0202490; Allocca et al. (2007) J. Virol. 81:11372; Boucas et al.(2009) J. Gene Med. 11:1103.

SUMMARY OF THE INVENTION

The present disclosure provides adeno-associated virus (AAV) virionswith altered capsid protein, where the AAV virions exhibit greaterinfectivity of a retinal cell, when administered via intravitrealinjection, compared to wild-type AAV. The present disclosure furtherprovides methods of delivering a gene product to a retinal cell in anindividual, and methods of treating ocular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a representative three-dimensional model of AAV2containing a random heptamer following amino acid 587.

FIG. 2 depicts greater levels of intravitreal transduction by AAV2 7M8variant (right), relative to AAV2 (left).

FIG. 3 provides representative fluorescence images of retinal cryoslicesshowing green fluorescent protein (GFP) expression resulting from 7M8carrying the GFP gene under the control of the ubiquitous CAG promoter(left) or a photoreceptor-specific Rho promoter (right).

FIG. 4 depicts GFP⁺ photoreceptor cells per million retinal cells ascounted by flow cytometry, following transduction by 7M8 or by 7M8bearing 4 tyrosine mutations (7m8.4YF).

FIG. 5 provides an amino acid sequence of AAV2 VP1 (SEQ ID NO:1).

FIG. 6 provides amino acid sequences corresponding to amino acids570-610 of AAV2 (FIG. 5) of AAV capsid protein VP1 of various AAVserotypes.

FIGS. 7A-7I depict structural improvements in the Rs1h−/− mouse retinaafter gene transfer.

FIGS. 8A-8D depict functional rescue of the electroretinogram 8A and 8Bwaves following RS1 gene delivery.

FIGS. 9A-9E depict sustained improvements in retinal thickness measuredat 10 months post 7m8-rho-RS1 treatment.

FIG. 10 provides an amino acid sequence of retinoschisin.

FIG. 11 provides an amino acid sequence of brain derived neurotrophicfactor.

FIG. 12 provides an amino acid sequence of RPE65.

FIGS. 13A-13C provide the nucleotide sequence of the 7m8-rho-RS1construct.

FIG. 14 provides an amino acid sequence of peripherin-2.

FIG. 15 provides an amino acid sequence of peripherin.

FIG. 16 provides an amino acid sequence of retinitis pigmentosa GTPaseregulator-interacting protein-1.

FIGS. 17A-17C provide an alignment of amino acid sequences of AAV capsidprotein loop IV (GH loop) regions. Insertion sites are shown in bold andunderlining.

FIGS. 18A-18C provide an alignment of amino acid sequences of AAV capsidprotein GH loop regions, with heterologous peptide insertions.

FIG. 19 provides a fluorescence fundus image showing GFP expression incentral primate retina 9 weeks after administration of 7m8 carrying GFPunder the control of a connexin36 promoter.

DEFINITIONS

The term “retinal cell” can refer herein to any of the cell types thatcomprise the retina, such as retinal ganglion cells, amacrine cells,horizontal cells, bipolar cells, and photoreceptor cells including rodsand cones, Müller glial cells, and retinal pigmented epithelium.

“AAV” is an abbreviation for adeno-associated virus, and may be used torefer to the virus itself or derivatives thereof. The term covers allsubtypes and both naturally occurring and recombinant forms, exceptwhere required otherwise. The abbreviation “rAAV” refers to recombinantadeno-associated virus, also referred to as a recombinant AAV vector (or“rAAV vector”). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2(AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAVtype 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV,bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, andovine AAV. “Primate AAV” refers to AAV that infect primates,“non-primate AAV” refers to AAV that infect non-primate mammals, “bovineAAV” refers to AAV that infect bovine mammals, etc.

The genomic sequences of various serotypes of AAV, as well as thesequences of the native terminal repeats (TRs), Rep proteins, and capsidsubunits are known in the art. Such sequences may be found in theliterature or in public databases such as GenBank. See, e.g., GenBankAccession Numbers NC_002077 (AAV-1), AF063497 (AAV-1), NC_001401(AAV-2), AF043303 (AAV-2), NC_001729 (AAV-3), NC_001829 (AAV-4), U89790(AAV-4), NC_006152 (AAV-5), AF513851 (AAV-7), AF513852 (AAV-8), andNC_006261 (AAV-8); the disclosures of which are incorporated byreference herein for teaching AAV nucleic acid and amino acid sequences.See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini etal. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology73:1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al.(1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208;Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat.Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383;international patent publications WO 00/28061, WO 99/61601, WO 98/11244;and U.S. Pat. No. 6,156,303.

An “rAAV vector” as used herein refers to an AAV vector comprising apolynucleotide sequence not of AAV origin (i.e., a polynucleotideheterologous to AAV), typically a sequence of interest for the genetictransformation of a cell. In general, the heterologous polynucleotide isflanked by at least one, and generally by two, AAV inverted terminalrepeat sequences (ITRs). The term rAAV vector encompasses both rAAVvector particles and rAAV vector plasmids. An rAAV vector may either besingle-stranded (ssAAV) or self-complementary (scAAV).

An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refersto a viral particle composed of at least one AAV capsid protein(typically by all of the capsid proteins of a wild-type AAV) and anencapsidated polynucleotide rAAV vector. If the particle comprises aheterologous polynucleotide (i.e. a polynucleotide other than awild-type AAV genome such as a transgene to be delivered to a mammaliancell), it is typically referred to as an “rAAV vector particle” orsimply an “rAAV vector”. Thus, production of rAAV particle necessarilyincludes production of rAAV vector, as such a vector is contained withinan rAAV particle.

“Packaging” refers to a series of intracellular events that result inthe assembly and encapsidation of an AAV particle.

AAV “rep” and “cap” genes refer to polynucleotide sequences encodingreplication and encapsidation proteins of adeno-associated virus. AAVrep and cap are referred to herein as AAV “packaging genes.”

A “helper virus” for AAV refers to a virus that allows AAV (e.g.wild-type AAV) to be replicated and packaged by a mammalian cell. Avariety of such helper viruses for AAV are known in the art, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand available from depositories such as the ATCC. Viruses of the herpesfamily include, for example, herpes simplex viruses (HSV) andEpstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) andpseudorabies viruses (PRV); which are also available from depositoriessuch as ATCC.

“Helper virus function(s)” refers to function(s) encoded in a helpervirus genome which allow AAV replication and packaging (in conjunctionwith other requirements for replication and packaging described herein).As described herein, “helper virus function” may be provided in a numberof ways, including by providing helper virus or providing, for example,polynucleotide sequences encoding the requisite function(s) to aproducer cell in trans. For example, a plasmid or other expressionvector comprising nucleotide sequences encoding one or more adenoviralproteins is transfected into a producer cell along with an rAAV vector.

An “infectious” virus or viral particle is one that comprises acompetently assembled viral capsid and is capable of delivering apolynucleotide component into a cell for which the viral species istropic. The term does not necessarily imply any replication capacity ofthe virus. Assays for counting infectious viral particles are describedelsewhere in this disclosure and in the art. Viral infectivity can beexpressed as the ratio of infectious viral particles to total viralparticles. Methods of determining the ratio of infectious viral particleto total viral particle are known in the art. See, e.g., Grainger et al.(2005) Mol. Ther. 11:5337 (describing a TCID50 infectious titer assay);and Zolotukhin et al. (1999) Gene Ther. 6:973. See also the Examples.

A “replication-competent” virus (e.g. a replication-competent AAV)refers to a phenotypically wild-type virus that is infectious, and isalso capable of being replicated in an infected cell (i.e. in thepresence of a helper virus or helper virus functions). In the case ofAAV, replication competence generally requires the presence offunctional AAV packaging genes. In general, rAAV vectors as describedherein are replication-incompetent in mammalian cells (especially inhuman cells) by virtue of the lack of one or more AAV packaging genes.Typically, such rAAV vectors lack any AAV packaging gene sequences inorder to minimize the possibility that replication competent AAV aregenerated by recombination between AAV packaging genes and an incomingrAAV vector. In many embodiments, rAAV vector preparations as describedherein are those which contain few if any replication competent AAV(rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10²rAAV particles, less than about 1 rcAAV per 10⁴ rAAV particles, lessthan about 1 rcAAV per 10⁸ rAAV particles, less than about 1 rcAAV per10¹² rAAV particles, or no rcAAV).

The term “polynucleotide” refers to a polymeric form of nucleotides ofany length, including deoxyribonucleotides or ribonucleotides, oranalogs thereof. A polynucleotide may comprise modified nucleotides,such as methylated nucleotides and nucleotide analogs, and may beinterrupted by non-nucleotide components. If present, modifications tothe nucleotide structure may be imparted before or after assembly of thepolymer. The term polynucleotide, as used herein, refers interchangeablyto double- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of twocomplementary single-stranded forms known or predicted to make up thedouble-stranded form.

Nucleic acid hybridization reactions can be performed under conditionsof different “stringency”. Conditions that increase stringency of ahybridization reaction of widely known and published in the art. See,e.g., Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989,herein incorporated by reference. For example, see page 7.52 of Sambrooket al. Examples of relevant conditions include (in order of increasingstringency): incubation temperatures of 25° C., 37° C., 50° C. and 68°C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where 1×SSCis 0.15 M NaCl and 15 mM citrate buffer) and their equivalents usingother buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%;incubation times from 5 minutes to 24 hours; 1, 2, or more washingsteps; wash incubation times of 1, 2, or 15 minutes; and wash solutionsof 6×SSC, 1×SSC, 0.1×SSC, or deionized water. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 1×SSC (150 mM NaCl, 15 mM sodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C. As another example,stringent hybridization conditions comprise: prehybridization for 8hours to overnight at 65° C. in a solution comprising 6× single strengthcitrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0),5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herringsperm DNA; hybridization for 18-20 hours at 65° C. in a solutioncontaining 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05%sodium pyrophosphate; and washing of filters at 65° C. for 1 h in asolution containing 0.2×SSC and 0.1% SDS (sodium dodecyl sulfate).

Stringent hybridization conditions are hybridization conditions that areat least as stringent as the above representative conditions. Otherstringent hybridization conditions are known in the art and may also beemployed to identify nucleic acids of this particular embodiment of theinvention.

“T_(m)” is the temperature in degrees Celsius at which 50% of apolynucleotide duplex made of complementary strands hydrogen bonded inanti-parallel direction by Watson-Crick base pairing dissociates intosingle strands under conditions of the experiment. T_(m) may bepredicted according to a standard formula, such as:

T _(m)=81.5+16.6 log [X ⁺]+0.41(% G/C)−0.61(% F)−600/L

where [X⁺] is the cation concentration (usually sodium ion, Na⁺) inmol/L; (% G/C) is the number of G and C residues as a percentage oftotal residues in the duplex; (% F) is the percent formamide in solution(wt/vol); and L is the number of nucleotides in each strand of theduplex.

A polynucleotide or polypeptide has a certain percent “sequenceidentity” to another polynucleotide or polypeptide, meaning that, whenaligned, that percentage of bases or amino acids are the same whencomparing the two sequences. Sequence similarity can be determined in anumber of different manners. To determine sequence identity, sequencescan be aligned using the methods and computer programs, including BLAST,available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Anotheralignment algorithm is FASTA, available in the Genetics Computing Group(GCG) package, from Madison, Wis., USA, a wholly owned subsidiary ofOxford Molecular Group, Inc. Other techniques for alignment aredescribed in Methods in Enzymology, vol. 266: Computer Methods forMacromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press,Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Ofparticular interest are alignment programs that permit gaps in thesequence. The Smith-Waterman is one type of algorithm that permits gapsin sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also,the GAP program using the Needleman and Wunsch alignment method can beutilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)

Of interest is the BestFit program using the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981) todetermine sequence identity. The gap generation penalty will generallyrange from 1 to 5, usually 2 to 4 and in many embodiments will be 3. Thegap extension penalty will generally range from about 0.01 to 0.20 andin many instances will be 0.10. The program has default parametersdetermined by the sequences inputted to be compared. Preferably, thesequence identity is determined using the default parameters determinedby the program. This program is available also from Genetics ComputingGroup (GCG) package, from Madison, Wis., USA.

Another program of interest is the FastDB algorithm. FastDB is describedin Current Methods in Sequence Comparison and Analysis, MacromoleculeSequencing and Synthesis, Selected Methods and Applications, pp.127-149, 1988, Alan R. Liss, Inc.

Percent sequence identity is calculated by FastDB based upon thefollowing parameters:

Mismatch Penalty: 1.00;

Gap Penalty: 1.00;

Gap Size Penalty: 0.33; and

Joining Penalty: 30.0.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular protein after beingtranscribed and translated.

A “gene product” is a molecule resulting from expression of a particulargene. Gene products include, e.g., a polypeptide, an aptamer, aninterfering RNA, an mRNA, and the like.

A “small interfering” or “short interfering RNA” or siRNA is a RNAduplex of nucleotides that is targeted to a gene interest (a “targetgene”). An “RNA duplex” refers to the structure formed by thecomplementary pairing between two regions of a RNA molecule. siRNA is“targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In some embodiments, the length of the duplex of siRNAsis less than 30 nucleotides. In some embodiments, the duplex can be 29,28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11or 10 nucleotides in length. In some embodiments, the length of theduplex is 19-25 nucleotides in length. The RNA duplex portion of thesiRNA can be part of a hairpin structure. In addition to the duplexportion, the hairpin structure may contain a loop portion positionedbetween the two sequences that form the duplex. The loop can vary inlength. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13nucleotides in length. The hairpin structure can also contain 3′ or 5′overhang portions. In some embodiments, the overhang is a 3′ or a 5′overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

A “short hairpin RNA,” or shRNA, is a polynucleotide construct that canbe made to express an interfering RNA such as siRNA.

“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules that contributes to thefunctional regulation of a polynucleotide, including replication,duplication, transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

“Operatively linked” or “operably linked” refers to a juxtaposition ofgenetic elements, wherein the elements are in a relationship permittingthem to operate in the expected manner. For instance, a promoter isoperatively linked to a coding region if the promoter helps initiatetranscription of the coding sequence. There may be intervening residuesbetween the promoter and coding region so long as this functionalrelationship is maintained.

An “expression vector” is a vector comprising a region which encodes apolypeptide of interest, and is used for effecting the expression of theprotein in an intended target cell. An expression vector also comprisescontrol elements operatively linked to the encoding region to facilitateexpression of the protein in the target. The combination of controlelements and a gene or genes to which they are operably linked forexpression is sometimes referred to as an “expression cassette,” a largenumber of which are known and available in the art or can be readilyconstructed from components that are available in the art.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. Thus, forexample, an rAAV that includes a heterologous nucleic acid encoding aheterologous gene product is an rAAV that includes a nucleic acid notnormally included in a naturally-occurring, wild-type AAV, and theencoded heterologous gene product is a gene product not normally encodedby a naturally-occurring, wild-type AAV.

The terms “genetic alteration” and “genetic modification” (andgrammatical variants thereof), are used interchangeably herein to referto a process wherein a genetic element (e.g., a polynucleotide) isintroduced into a cell other than by mitosis or meiosis. The element maybe heterologous to the cell, or it may be an additional copy or improvedversion of an element already present in the cell. Genetic alterationmay be effected, for example, by transfecting a cell with a recombinantplasmid or other polynucleotide through any process known in the art,such as electroporation, calcium phosphate precipitation, or contactingwith a polynucleotide-liposome complex. Genetic alteration may also beeffected, for example, by transduction or infection with a DNA or RNAvirus or viral vector. Generally, the genetic element is introduced intoa chromosome or mini-chromosome in the cell; but any alteration thatchanges the phenotype and/or genotype of the cell and its progeny isincluded in this term.

A cell is said to be “stably” altered, transduced, genetically modified,or transformed with a genetic sequence if the sequence is available toperform its function during extended culture of the cell in vitro.Generally, such a cell is “heritably” altered (genetically modified) inthat a genetic alteration is introduced which is also inheritable byprogeny of the altered cell.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The terms also encompass an amino acid polymer that has beenmodified; for example, disulfide bond formation, glycosylation,lipidation, phosphorylation, or conjugation with a labeling component.Polypeptides such as anti-angiogenic polypeptides, neuroprotectivepolypeptides, and the like, when discussed in the context of deliveringa gene product to a mammalian subject, and compositions therefor, referto the respective intact polypeptide, or any fragment or geneticallyengineered derivative thereof, which retains the desired biochemicalfunction of the intact protein. Similarly, references to nucleic acidsencoding anti-angiogenic polypeptides, nucleic acids encodingneuroprotective polypeptides, and other such nucleic acids for use indelivery of a gene product to a mammalian subject (which may be referredto as “transgenes” to be delivered to a recipient cell), includepolynucleotides encoding the intact polypeptide or any fragment orgenetically engineered derivative possessing the desired biochemicalfunction.

An “isolated” plasmid, nucleic acid, vector, virus, virion, host cell,or other substance refers to a preparation of the substance devoid of atleast some of the other components that may also be present where thesubstance or a similar substance naturally occurs or is initiallyprepared from. Thus, for example, an isolated substance may be preparedby using a purification technique to enrich it from a source mixture.Enrichment can be measured on an absolute basis, such as weight pervolume of solution, or it can be measured in relation to a second,potentially interfering substance present in the source mixture.Increasing enrichments of the embodiments of this disclosure areincreasingly more isolated. An isolated plasmid, nucleic acid, vector,virus, host cell, or other substance is in some embodiments purified,e.g., from about 80% to about 90% pure, at least about 90% pure, atleast about 95% pure, at least about 98% pure, or at least about 99%, ormore, pure.

As used herein, the terms “treatment,” “treating,” and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment,” as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease or at risk of acquiring the diseasebut has not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; and (c) relieving the disease, i.e.,causing regression of the disease.

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to a mammal, including, but notlimited to, human and non-human primates, including simians and humans;mammalian sport animals (e.g., horses); mammalian farm animals (e.g.,sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents(e.g., mice, rats, etc.).

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “arecombinant AAV virion” includes a plurality of such virions andreference to “the photoreceptor cell” includes reference to one or morephotoreceptor cells and equivalents thereof known to those skilled inthe art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides adeno-associated virus (AAV) virionswith altered capsid protein, where the AAV virions exhibit greaterinfectivity of a retinal cell, when administered via intravitrealinjection, compared to wild-type AAV when administered via intravitrealinjection. The present disclosure further provides methods of deliveringa gene product to a retinal cell in an individual, and methods oftreating ocular disease.

The retinal cell can be a photoreceptor (e.g., rods; cones), a retinalganglion cell (RGC), a Müller cell (a Müller glial cell), a bipolarcell, an amacrine cell, a horizontal cell, or a retinal pigmentedepithelium (RPE) cell.

Variant AAV Capsid Polypeptides

The present disclosure provides a variant AAV capsid protein, where thevariant AAV capsid protein comprises an insertion of from about 5 aminoacids to about 11 amino acids in an insertion site in the capsid proteinGH loop or loop IV, relative to a corresponding parental AAV capsidprotein, and where the variant capsid protein, when present in an AAVvirion, confers increased infectivity of a retinal cell compared to theinfectivity of the retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein. In some cases, the retinalcell is a photoreceptor cell (e.g., rods; cones). In other cases, theretinal cell is an RGC. In other cases, the retinal cell is an RPE cell.In other cases, the retinal cell is a Müller cell. Other retinal cellsinclude amacrine cells, bipolar cells, and horizontal cells. An“insertion of from about 5 amino acids to about 11 amino acids” is alsoreferred to herein as a “peptide insertion” (e.g., a heterologouspeptide insertion). A “corresponding parental AAV capsid protein” refersto an AAV capsid protein of the same AAV serotype, without the peptideinsertion.

The insertion site is in the GH loop, or loop IV, of the AAV capsidprotein, e.g., in a solvent-accessible portion of the GH loop, or loopIV, of the AAV capsid protein. For the GH loop/loop IV of AAV capsid,see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809; Padron et al.(2005) J. Virol. 79:5047; and Shen et al. (2007) Mol. Ther. 15:1955. Forexample, the insertion site can be within amino acids 411-650 of an AAVcapsid protein, as depicted in FIGS. 17A and 17B. For example, theinsertion site can be within amino acids 570-611 of AAV2, within aminoacids 571-612 of AAV1, within amino acids 560-601 of AAV5, within aminoacids 571 to 612 of AAV6, within amino acids 572 to 613 of AAV7, withinamino acids 573 to 614 of AAV8, within amino acids 571 to 612 of AAV9,or within amino acids 573 to 614 of AAV10, as depicted in FIG. 6.

In some cases, from about 5 amino acids to about 11 amino acids areinserted in an insertion site in the GH loop or loop IV of the capsidprotein relative to a corresponding parental AAV capsid protein. Forexample, the insertion site can be between amino acids 587 and 588 ofAAV2, or the corresponding positions of the capsid subunit of anotherAAV serotype. It should be noted that the insertion site 587/588 isbased on an AAV2 capsid protein. From about 5 amino acids to about 11amino acids can be inserted in a corresponding site in an AAV serotypeother than AAV2 (e.g., AAV8, AAV9, etc.). Those skilled in the art wouldknow, based on a comparison of the amino acid sequences of capsidproteins of various AAV serotypes, where an insertion site“corresponding to amino acids 587-588 of AAV2” would be in a capsidprotein of any given AAV serotype. Sequences corresponding to aminoacids 570-611 of capsid protein VP1 of AAV2 (see FIG. 5) in various AAVserotypes are shown in FIG. 6. See, e.g., GenBank Accession No.NP_049542 for AAV1; GenBank Accession No. AAD13756 for AAV5; GenBankAccession No. AAB95459 for AAV6; GenBank Accession No. YP_077178 forAAV7; GenBank Accession No. YP_077180 for AAV8; GenBank Accession No.AAS99264 for AAV9 and GenBank Accession No. AAT46337 for AAV10.

In some embodiments, the insertion site is a single insertion sitebetween two adjacent amino acids located between amino acids 570-614 ofVP1 of any AAV serotype, e.g., the insertion site is between twoadjacent amino acids located in amino acids 570-610, amino acids580-600, amino acids 570-575, amino acids 575-580, amino acids 580-585,amino acids 585-590, amino acids 590-600, or amino acids 600-614, of VP1of any AAV serotype or variant. For example, the insertion site can bebetween amino acids 580 and 581, amino acids 581 and 582, amino acids583 and 584, amino acids 584 and 585, amino acids 585 and 586, aminoacids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, oramino acids 589 and 590. The insertion site can be between amino acids575 and 576, amino acids 576 and 577, amino acids 577 and 578, aminoacids 578 and 579, or amino acids 579 and 580. The insertion site can bebetween amino acids 590 and 591, amino acids 591 and 592, amino acids592 and 593, amino acids 593 and 594, amino acids 594 and 595, aminoacids 595 and 596, amino acids 596 and 597, amino acids 597 and 598,amino acids 598 and 599, or amino acids 599 and 600.

For example, the insertion site can be between amino acids 587 and 588of AAV2, between amino acids 590 and 591 of AAV1, between amino acids575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, betweenamino acids 589 and 590 of AAV7, between amino acids 590 and 591 ofAAV8, between amino acids 588 and 589 of AAV9, or between amino acids588 and 589 of AAV10.

As another example, the insertion site can be between amino acids 450and 460 of an AAV capsid protein, as shown in FIG. 17A. For example, theinsertion site can be at (e.g., immediately N-terminal to) amino acid453 of AAV2, at amino acid 454 of AAV1, at amino acid 454 of AAV6, atamino acid 456 of AAV7, at amino acid 456 of AAV8, at amino acid 454 ofAAV9, or at amino acid 456 of AAV10, as shown in FIG. 17A.

In some embodiments, a subject capsid protein includes a GH loopcomprising an amino acid sequence having at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, at least about 99%,or 100%, amino acid sequence identity to an amino acid sequence setforth in FIG. 18A-C.

Insertion Peptides

As noted above, a peptide of from about 5 amino acids to about 11 aminoacids in length is inserted into the GH loop of an AAV capsid. Theinsertion peptide has a length of 5 amino acids, 6 amino acids, 7 aminoacids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids.

The insertion peptide can comprise an amino acid sequence of any one ofthe formulas set forth below.

For example, an insertion peptide can be a peptide of from 5 to 11 aminoacids in length, where the insertion peptide is of Formula I:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is selected from Leu, Asn, and Lys;

X₂ is selected from Gly, Glu, Ala, and Asp;

X₃ is selected from Glu, Thr, Gly, and Pro;

X₄ is selected from Thr, Ile, Gln, and Lys;

X₅ is selected from Thr and Ala;

X₆ is selected from Arg, Asn, and Thr;

X₇, if present, is selected from Pro and Asn.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula Ha:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

each of X₁-X₄ is any amino acid;

X₅ is Thr;

X₆ is Arg; and

X₇ is Pro.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula IIb:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is selected from Leu and Asn;

X₂, if present, is selected from Gly and Glu;

X₃ is selected from Glu and Thr;

X₄ is selected from Thr and Ile;

X₅ is Thr;

X₆ is Arg; and

X₇ is Pro.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula II:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is Lys;

X₂ is selected from Ala and Asp;

X₃ is selected from Gly and Pro;

X₄ is selected from Gln and Lys;

X₅ is selected from Thr and Ala;

X₆ is selected from Asn and Thr;

X₇, if present, is Asn.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula IV:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is a positively charged amino acid or an uncharged aminoacid; or is selected from Leu, Asn, Arg, Ala, Ser, and Lys;

X₂ is a negatively charged amino acid or an uncharged amino acid; or isselected from Gly, Glu, Ala, Val, Thr, and Asp;

X₃ is a negatively charged amino acid or an uncharged amino acid; or isselected from Glu, Thr, Gly, Asp, or Pro;

X₄ is selected from Thr, Ile, Gly, Lys, Asp, and Gln;

X₅ is a polar amino acid, an alcohol (an amino acid having a freehydroxyl group), or a hydrophobic amino acid; or is selected from Thr,Ser, Val, and Ala;

X₆ is a positively charged amino acid or an uncharged amino acid; or isselected from Arg, Val, Lys, Pro, Thr, and Asn; and

X₇, if present, is a positively charged amino acid or an uncharged aminoacid; or is selected from Pro, Gly, Phe, Asn, and Arg.

As non-limiting examples, the insertion peptide can comprise an aminoacid sequence selected from LGETTRP (SEQ ID NO:13), NETITRP (SEQ IDNO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTR (SEQ IDNO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQID NO:60).

In some cases, the insertion peptide has from 1 to 4 spacer amino acids(Y₁—Y₄) at the amino terminus and/or at the carboxyl terminus of any oneof LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ IDNO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ IDNO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60). Suitablespacer amino acids include, but are not limited to, leucine, alanine,glycine, and serine.

For example, in some cases, an insertion peptide has one of thefollowing amino acid sequences: LALGETTRPA (SEQ ID NO:45); LANETITRPA(SEQ ID NO:46), LAKAGQANNA (SEQ ID NO:47), LAKDPKTTNA (SEQ ID NO:48),LAKDTDTTRA (SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA (SEQ IDNO:63), and LASTGKVPNA (SEQ ID NO:64). As another example, in somecases, an insertion peptide has one of the following amino acidsequences: AALGETTRPA (SEQ ID NO:49); AANETITRPA (SEQ ID NO:50),AAKAGQANNA (SEQ ID NO:51), and AAKDPKTTNA (SEQ ID NO:52). As yet anotherexample, in some cases, an insertion peptide has one of the followingamino acid sequences: GLGETTRPA (SEQ ID NO:53); GNETITRPA (SEQ IDNO:54), GKAGQANNA (SEQ ID NO:55), and GKDPKTTNA (SEQ ID NO:56). Asanother example, in some cases, an insertion peptide comprises one ofKDTDTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59),and STGKVPN (SEQ ID NO:60), flanked on the C-terminus by AA and on theN-terminus by A; or comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG(SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60)flanked on the C-terminus by G and on the N-terminus by A.

In some embodiments, a subject variant AAV capsid does not include anyother amino acid substitutions, insertions, or deletions, other than aninsertion of from about 5 amino acids to about 11 amino acids in the GHloop or loop IV relative to a corresponding parental AAV capsid protein.In other embodiments, a subject variant AAV capsid includes from 1 toabout 25 amino acid insertions, deletions, or substitutions, compared tothe parental AAV capsid protein, in addition to an insertion of fromabout 5 amino acids to about 11 amino acids in the GH loop or loop IVrelative to a corresponding parental AAV capsid protein. For example, insome embodiments, a subject variant AAV capsid includes from 1 to about5, from about 5 to about 10, from about 10 to about 15, from about 15 toabout 20, or from about 20 to about 25 amino acid insertions, deletions,or substitutions, compared to the parental AAV capsid protein, inaddition to an insertion of from about 5 amino acids to about 11 aminoacids in the GH loop or loop IV relative to a corresponding parental AAVcapsid protein.

In some embodiments, a subject variant capsid polypeptide does notinclude one, two, three, or four, of the following amino acidsubstitutions: Y273F, Y444F, Y500F, and Y730F.

In some embodiments, a subject variant capsid polypeptide comprises, inaddition to an insertion peptide as described above, one, two, three, orfour, of the following amino acid substitutions: Y273F, Y444F, Y500F,and Y730F.

In some embodiments, a variant AAV capsid polypeptide is a chimericcapsid, e.g., the capsid comprises a portion of an AAV capsid of a firstAAV serotype and a portion of an AAV capsid of a second serotype; andcomprises an insertion of from about 5 amino acids to about 11 aminoacids in the GH loop or loop IV relative to a corresponding parental AAVcapsid protein.

In some embodiments, a subject variant capsid protein comprises an aminoacid sequence having at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, or at least about 99%, amino acidsequence identity to the amino acid sequence provided in FIG. 5; and aninsertion of from about 5 amino acids to about 11 amino acids in the GHloop or loop IV relative to a corresponding parental AAV capsid protein.

In some embodiments, a subject variant capsid protein is isolated, e.g.,purified. In some cases, a subject variant capsid protein is included inan AAV vector, which is also provided. As described in detail below, asubject variant capsid protein can be included in a recombinant AAVvirion.

Recombinant AAV Virion

The present disclosure provides a recombinant adeno-associated virus(rAAV) virion comprising: a) a variant AAV capsid protein, where thevariant AAV capsid protein comprises an insertion of from about 5 aminoacids to about 11 amino acids in an insertion site in the capsid proteinGH loop or loop IV, relative to a corresponding parental AAV capsidprotein, and where the variant capsid protein confers increasedinfectivity of a retinal cell compared to the infectivity of the retinalcell by an AAV virion comprising the corresponding parental AAV capsidprotein; and b) a heterologous nucleic acid comprising a nucleotidesequence encoding a gene product. In some cases, the retinal cell is aphotoreceptor cell (e.g., rods and/or cones). In other cases, theretinal cell is an RGC cell. In other cases, the retinal cell is an RPEcell. In other cases, the retinal cell is a Müller cell. In other cases,retinal cells may include amacrine cells, bipolar cells, and horizontalcells. An “insertion of from about 5 amino acids to about 11 aminoacids” is also referred to herein as a “peptide insertion” (e.g., aheterologous peptide insertion). A “corresponding parental AAV capsidprotein” refers to an AAV capsid protein of the same AAV serotype,without the peptide insertion.

The insertion site is in the GH loop, or loop IV, of the AAV capsidprotein, e.g., in a solvent-accessible portion of the GH loop, or loopIV, of the AAV capsid protein. For the GH loop, see, e.g., van Vliet etal. (2006) Mol. Ther. 14:809; Padron et al. (2005) J. Virol. 79:5047;and Shen et al. (2007) Mol. Ther. 15:1955. For example, the insertionsite is within amino acids 570-611 of AAV2, within amino acids 571-612of AAV1, within amino acids 560-601 of AAV5, within amino acids 571 to612 of AAV6, within amino acids 572 to 613 of AAV7, within amino acids573 to 614 of AAV8, within amino acids 571 to 612 of AAV9, or withinamino acids 573 to 614 of AAV10.

From about 5 amino acids to about 11 amino acids are inserted in aninsertion site in the GH loop or loop IV of the capsid protein relativeto a corresponding parental AAV capsid protein. For example, theinsertion site can be between amino acids 587 and 588 of AAV2, or thecorresponding positions of the capsid subunit of another AAV serotype.It should be noted that the insertion site 587/588 is based on an AAV2capsid protein. From about 5 amino acids to about 11 amino acids can beinserted in a corresponding site in an AAV serotype other than AAV2(e.g., AAV8, AAV9, etc.). Those skilled in the art would know, based ona comparison of the amino acid sequences of capsid proteins of variousAAV serotypes, where an insertion site “corresponding to amino acids587-588 of AAV2” would be in a capsid protein of any given AAV serotype.Sequences corresponding to amino acids 570-611 of capsid protein VP1 ofAAV2 (see FIG. 5) in various AAV serotypes are shown in FIG. 6.

In some embodiments, the insertion site is a single insertion sitebetween two adjacent amino acids located between amino acids 570-614 ofVP1 of any AAV serotype, e.g., the insertion site is between twoadjacent amino acids located in amino acids 570-614, amino acids580-600, amino acids 570-575, amino acids 575-580, amino acids 580-585,amino acids 585-590, amino acids 590-600, or amino acids 600-610, of VP1of any AAV serotype or variant. For example, the insertion site can bebetween amino acids 580 and 581, amino acids 581 and 582, amino acids583 and 584, amino acids 584 and 585, amino acids 585 and 586, aminoacids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, oramino acids 589 and 590. The insertion site can be between amino acids575 and 576, amino acids 576 and 577, amino acids 577 and 578, aminoacids 578 and 579, or amino acids 579 and 580. The insertion site can bebetween amino acids 590 and 591, amino acids 591 and 592, amino acids592 and 593, amino acids 593 and 594, amino acids 594 and 595, aminoacids 595 and 596, amino acids 596 and 597, amino acids 597 and 598,amino acids 598 and 599, or amino acids 599 and 600.

For example, the insertion site can be between amino acids 587 and 588of AAV2, between amino acids 590 and 591 of AAV1, between amino acids575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, betweenamino acids 589 and 590 of AAV7, between amino acids 590 and 591 ofAAV8, between amino acids 588 and 589 of AAV9, or between amino acids589 and 590 of AAV10.

Insertion Peptides

As noted above, a subject rAAV virion comprises a peptide of from about5 amino acids to about 11 amino acids in length inserted into the GHloop of the AAV capsid. The insertion peptide has a length of 5 aminoacids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10amino acids, or 11 amino acids.

The insertion peptide can comprise an amino acid sequence of any one ofthe formulas set forth below.

For example, an insertion peptide can be a peptide of from 5 to 11 aminoacids in length, where the insertion peptide is of Formula I:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is selected from Leu, Asn, and Lys;

X₂ is selected from Gly, Glu, Ala, and Asp;

X₃ is selected from Glu, Thr, Gly, and Pro;

X₄ is selected from Thr, lie, Gin, and Lys;

X₅ is selected from Thr and Ala;

X₆ is selected from Arg, Asn, and Thr;

X₇, if present, is selected from Pro and Asn.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula Ha:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

each of X₁-X₄ is any amino acid;

X₅ is Thr;

X₆ is Arg; and

X₇ is Pro.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula IIb:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is selected from Leu and Asn;

X₂, if present, is selected from Gly and Glu;

X₃ is selected from Glu and Thr;

X₄ is selected from Thr and Ile;

X₅ is Thr;

X₆ is Arg; and

X₇ is Pro.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula II:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is Lys;

X₂ is selected from Ala and Asp;

X₃ is selected from Gly and Pro;

X₄ is selected from Gin and Lys;

X₅ is selected from Thr and Ala;

X₆ is selected from Asn and Thr;

X₇, if present, is Asn.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula IV:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is a positively charged amino acid or an uncharged aminoacid; or is selected from Leu, Asn, Arg, Ala, Ser, and Lys;

X₂ is a negatively charged amino acid or an uncharged amino acid; or isselected from Gly, Glu, Ala, Val, Thr, and Asp;

X₃ is a negatively charged amino acid or an uncharged amino acid; or isselected from Glu, Thr, Gly, Asp, or Pro;

X₄ is selected from Thr, Ile, Gly, Lys, Asp, and Gln;

X₅ is a polar amino acid, an alcohol (an amino acid having a freehydroxyl group), or a hydrophobic amino acid; or is selected from Thr,Ser, Val, and Ala;

X₆ is a positively charged amino acid or an uncharged amino acid; or isselected from Arg, Val, Lys, Pro, Thr, and Asn; and

X₇, if present, is a positively charged amino acid or an uncharged aminoacid; or is selected from Pro, Gly, Phe, Asn, and Arg.

As non-limiting examples, the insertion peptide can comprise an aminoacid sequence selected from LGETRP (SEQ ID NO:13), NETITRP (SEQ IDNO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ IDNO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQID NO:60).

In some cases, the insertion peptide has from 1 to 4 spacer amino acids(Y₁—Y₄) at the amino terminus and/or at the carboxyl terminus of any oneof LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ IDNO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ IDNO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60). Suitablespacer amino acids include, but are not limited to, leucine, alanine,glycine, and serine.

For example, in some cases, an insertion peptide has one of thefollowing amino acid sequences: LALGETTRPA (SEQ ID NO:45); LANETITRPA(SEQ ID NO:46), LAKAGQANNA (SEQ ID NO:47), LAKDPKTTNA (SEQ ID NO:48),LAKDTDTTRA (SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA (SEQ IDNO:63), and LASTGKVPNA (SEQ ID NO:64). As another example, in somecases, an insertion peptide has one of the following amino acidsequences: AALGETTRPA (SEQ ID NO:49); AANETITRPA (SEQ ID NO:50),AAKAGQANNA (SEQ ID NO:51), and AAKDPKTTNA (SEQ ID NO:52). As yet anotherexample, in some cases, an insertion peptide has one of the followingamino acid sequences: GLGETTRPA (SEQ ID NO:53); GNETITRPA (SEQ IDNO:54), GKAGQANNA (SEQ ID NO:55), and GKDPKTTNA (SEQ ID NO:56). Asanother example, in some cases, an insertion peptide comprises one ofKDTDTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59),and STGKVPN (SEQ ID NO:60), flanked on the C-terminus by AA and on theN-terminus by A; or comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG(SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60)flanked on the C-terminus by G and on the N-terminus by A.

In some embodiments, a subject rAAV virion capsid does not include anyother amino acid substitutions, insertions, or deletions, other than aninsertion of from about 7 amino acids to about 10 amino acids in the GHloop or loop IV relative to a corresponding parental AAV capsid protein.In other embodiments, a subject rAAV virion capsid includes from 1 toabout 25 amino acid insertions, deletions, or substitutions, compared tothe parental AAV capsid protein, in addition to an insertion of fromabout 7 amino acids to about 10 amino acids in the GH loop or loop IVrelative to a corresponding parental AAV capsid protein. For example, insome embodiments, a subject rAAV virion capsid includes from 1 to about5, from about 5 to about 10, from about 10 to about 15, from about 15 toabout 20, or from about 20 to about 25 amino acid insertions, deletions,or substitutions, compared to the parental AAV capsid protein, inaddition to an insertion of from about 7 amino acids to about 10 aminoacids in the GH loop or loop IV relative to a corresponding parental AAVcapsid protein.

In some embodiments, a subject rAAV virion capsid does not include one,two, three, or four, of the following amino acid substitutions: Y273F,Y444F, Y500F, and Y730F.

In some embodiments, a subject variant capsid polypeptide comprises, inaddition to an insertion peptide as described above, one, two, three, orfour, of the following amino acid substitutions: Y273F, Y444F, Y500F,and Y730F.

In some embodiments, a subject rAAV virion capsid is a chimeric capsid,e.g., the capsid comprises a portion of an AAV capsid of a first AAVserotype and a portion of an AAV capsid of a second serotype; andcomprises an insertion of from about 5 amino acids to about 11 aminoacids in the GH loop or loop IV relative to a corresponding parental AAVcapsid protein.

In some embodiments, a subject rAAV virion comprises a capsid proteincomprising an amino acid sequence having at least about 85%, at leastabout 90%, at least about 95%, at least about 98%, or at least about99%, amino acid sequence identity to the amino acid sequence provided inFIG. 5; and an insertion of from about 5 amino acids to about 11 aminoacids in the GH loop or loop IV relative to a corresponding parental AAVcapsid protein.

In some embodiments, a subject rAAV virion comprises a capsid proteinthat includes a GH loop comprising an amino acid sequence having atleast about 85%, at least about 90%, at least about 95%, at least about98%, at least about 99%, or 100%, amino acid sequence identity to anamino acid sequence set forth in FIG. 18A-C.

A subject rAAV virion exhibits at least 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold,increased infectivity of a retinal cell, compared to the infectivity ofthe retinal cell by an AAV virion comprising the corresponding parentalAAV capsid protein.

In some cases, a subject rAAV virion exhibits at least 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or morethan 50-fold, increased infectivity of a retinal cell, when administeredvia intravitreal injection, compared to the infectivity of the retinalcell by an AAV virion comprising the corresponding parental AAV capsidprotein, when administered via intravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a photoreceptor (rod orcone) cell, compared to the infectivity of the photoreceptor cell by anAAV virion comprising the corresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a photoreceptor (rod orcone) cell, when administered via intravitreal injection, compared tothe infectivity of the photoreceptor cell by an AAV virion comprisingthe corresponding parental AAV capsid protein, when administered viaintravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of an RGC, compared to theinfectivity of the RGC by an AAV virion comprising the correspondingparental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of an RGC, when administeredvia intravitreal injection, compared to the infectivity of the RGC by anAAV virion comprising the corresponding parental AAV capsid protein,when administered via intravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of an RPE cell, compared to theinfectivity of the RPE cell by an AAV virion comprising thecorresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of an RPE cell, whenadministered via intravitreal injection, compared to the infectivity ofthe RPE cell by an AAV virion comprising the corresponding parental AAVcapsid protein, when administered via intravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a Müller cell, compared tothe infectivity of the Müller cell by an AAV virion comprising thecorresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a Müller cell, whenadministered via intravitreal injection, compared to the infectivity ofthe Müller cell by an AAV virion comprising the corresponding parentalAAV capsid protein, when administered via intravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a bipolar cell, compared tothe infectivity of the bipolar cell by an AAV virion comprising thecorresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a bipolar cell, whenadministered via intravitreal injection, compared to the infectivity ofthe bipolar cell by an AAV virion comprising the corresponding parentalAAV capsid protein, when administered via intravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of an amacrine cell, comparedto the infectivity of the amacrine cell by an AAV virion comprising thecorresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of an amacrine cell, whenadministered via intravitreal injection, compared to the infectivity ofthe amacrine cell by an AAV virion comprising the corresponding parentalAAV capsid protein, when administered via intravitreal injection.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a horizontal cell, comparedto the infectivity of the horizontal cell by an AAV virion comprisingthe corresponding parental AAV capsid protein.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a horizontal cell, whenadministered via intravitreal injection, compared to the infectivity ofthe horizontal cell by an AAV virion comprising the correspondingparental AAV capsid protein, when administered via intravitrealinjection.

In some cases, a subject rAAV virion exhibits at least 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or morethan 50-fold, increased ability to cross the internal limiting membrane(ILM), compared to the ability of an AAV virion comprising thecorresponding parental AAV capsid protein to cross the ILM.

In some cases, a subject rAAV virion exhibits at least 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or morethan 50-fold, increased ability, when administered via intravitrealinjection, to cross the internal limiting membrane (ILM), compared tothe ability of an AAV virion comprising the corresponding parental AAVcapsid protein to cross the ILM when administered via intravitrealinjection.

A subject rAAV virion can cross the ILM, and can also traverse celllayers, including Müller cells, amacrine cells, etc., to reach thephotoreceptor cells and or RPE cells. For example, a subject rAAVvirion, when administered via intravitreal injection, can cross the ILM,and can also traverse cell layers, including Müller cells, amacrinecells, etc., to reach the photoreceptor cells and or RPE cells.

In some embodiments, a subject rAAV virion selectively infects a retinalcell, e.g., a subject rAAV virion infects a retinal cell with 10-fold,15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificitythan a non-retinal cell, e.g., a cell outside the eye. For example, insome embodiments, a subject rAAV virion selectively infects a retinalcell, e.g., a subject rAAV virion infects a photoreceptor cell with10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold,specificity than a non-retinal cell, e.g., a cell outside the eye.

In some embodiments, a subject rAAV virion selectively infects aphotoreceptor cell, e.g., a subject rAAV virion infects a photoreceptorcell with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than50-fold, specificity than a non-photoreceptor cell present in the eye,e.g., a retinal ganglion cell, a Müller cell, etc.

In some embodiments, a subject rAAV virion exhibits at least 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, ormore than 50-fold, increased infectivity of a photoreceptor cell, whenadministered via intravitreal injection, compared to the infectivity ofthe photoreceptor cell by an AAV virion comprising the correspondingparental AAV capsid protein, when administered via intravitrealinjection.

Gene Products

A subject rAAV virion comprises a heterologous nucleic acid comprising anucleotide sequence encoding a gene product. In some embodiments, thegene product is an interfering RNA. In some embodiments, the geneproduct is an aptamer. In some embodiments, the gene product is apolypeptide. In some embodiments, the gene product is a site-specificnuclease that provide for site-specific knock-down of gene function.

Interfering RNA

Where the gene product is an interfering RNA (RNAi), suitable RNAiinclude RNAi that decrease the level of an apoptotic or angiogenicfactor in a cell. For example, an RNAi can be an shRNA or siRNA thatreduces the level of a gene product that induces or promotes apoptosisin a cell. Genes whose gene products induce or promote apoptosis arereferred to herein as “pro-apoptotic genes” and the products of thosegenes (mRNA; protein) are referred to as “pro-apoptotic gene products.”Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad geneproducts. See, e.g., U.S. Pat. No. 7,846,730.

Interfering RNAs could also be against an angiogenic product, forexample VEGF (e.g., Cand5; see, e.g., U.S. Patent Publication No.2011/0143400; U.S. Patent Publication No. 2008/0188437; and Reich et al.(2003) Mol. Vis. 9:210), VEGFR1 (e.g., Sirna-027; see, e.g., Kaiser etal. (2010) Am. J. Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther.13:225), or VEGFR2 (Kou et al. (2005) Biochem. 44:15064). See also, U.S.Pat. Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736; andU.S. Pat. Nos. 7,947,659 and 7,919,473.

Aptamers

Where the gene product is an aptamer, exemplary aptamers of interestinclude an aptamer against vascular endothelial growth factor (VEGF).See, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee etal. (2005) Proc. Natl. Acad. Sci. USA 102:18902. For example, a VEGFaptamer can comprise the nucleotide sequence5′-cgcaaucagugaaugcuuauacauccg-3′ (SEQ ID NO:17). Also suitable for useis a PDGF-specific aptamer, e.g., E10030; see, e.g., Ni and Hui (2009)Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol.207:407).

Polypeptides

Where the gene product is a polypeptide, the polypeptide is generally apolypeptide that enhances function of a retinal cell, e.g., the functionof a rod or cone photoreceptor cell, a retinal ganglion cell, a Müllercell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinalpigmented epithelial cell. Exemplary polypeptides includeneuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN);anti-angiogenic polypeptides (e.g., a soluble vascular endothelialgrowth factor (VEGF) receptor; a VEGF-binding antibody; a VEGF-bindingantibody fragment (e.g., a single chain anti-VEGF antibody); endostatin;tumstatin; angiostatin; a soluble Flt polypeptide (Lai et al. (2005)Mol. Ther. 12:659); an Fc fusion protein comprising a soluble Fltpolypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16:10); pigmentepithelium-derived factor (PEDF); a soluble Tie-2 receptor; etc.);tissue inhibitor of metalloproteinases-3 (TIMP-3); a light-responsiveopsin, e.g., a rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2,Bcl-Xl); and the like. Suitable polypeptides include, but are notlimited to, glial derived neurotrophic factor (GDNF); fibroblast growthfactor 2; neurturin (NTN); ciliary neurotrophic factor (CNTF); nervegrowth factor (NGF); neurotrophin-4 (NT4); brain derived neurotrophicfactor (BDNF; e.g., a polypeptide comprising an amino acid sequencehaving at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity to a contiguousstretch of from about 200 amino acids to 247 amino acids of the aminoacid sequence depicted in FIG. 11 (SEQ ID NO: 11)); epidermal growthfactor; rhodopsin; X-linked inhibitor of apoptosis; and Sonic hedgehog.

Suitable light-responsive opsins include, e.g., a light-responsive opsinas described in U.S. Patent Publication No. 2007/0261127 (e.g., ChR2;Chop2); U.S. Patent Publication No. 2001/0086421; U.S. PatentPublication No. 2010/0015095; and Diester et al. (2011) Nat. Neurosci.14:387.

Suitable polypeptides also include retinoschisin (e.g., a polypeptidecomprising an amino acid sequence having at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to a contiguous stretch of from about 200 amino acidsto 224 amino acids of the amino acid sequence depicted in FIG. 10 (SEQID NO: 10). Suitable polypeptides include, e.g., retinitis pigmentosaGTPase regulator (RGPR)-interacting protein-1 (see, e.g., GenBankAccession Nos. Q96KN7, Q9EPQ2, and Q9GLM3) (e.g., a polypeptidecomprising an amino acid sequence having at least about 90%, at leastabout 95%, at least about 98%, at least about 99%, or 100%, amino acidsequence identity to a contiguous stretch of from about 1150 amino acidsto about 1200 amino acids, or from about 1200 amino acids to 1286 aminoacids, of the amino acid sequence depicted in FIG. 16 (SEQ ID NO:21);peripherin-2 (Prph2) (see, e.g., GenBank Accession No. NP_000313 (e.g.,a polypeptide comprising an amino acid sequence having at least about90%, at least about 95%, at least about 98%, at least about 99%, or100%, amino acid sequence identity to a contiguous stretch of from about300 amino acids to 346 amino acids of the amino acid sequence depictedin FIG. 14 (SEQ ID NO:19); and Travis et al. (1991) Genomics 10:733);peripherin (e.g., a polypeptide comprising an amino acid sequence havingat least about 90%, at least about 95%, at least about 98%, at leastabout 99%, or 100%, amino acid sequence identity to a contiguous stretchof from about 400 amino acids to about 470 amino acids of the amino acidsequence depicted in FIG. 15 (SEQ ID NO:20); a retinal pigmentepithelium-specific protein (RPE65), (e.g., a polypeptide comprising anamino acid sequence having at least about 90%, at least about 95%, atleast about 98%, at least about 99%, or 100%, amino acid sequenceidentity to a contiguous stretch of from about 200 amino acids to 247amino acids of the amino acid sequence depicted in FIG. 12 (SEQ IDNO:12)) (see, e.g., GenBank AAC39660; and Morimura et al. (1998) Proc.Natl. Acad. Sci. USA 95:3088); and the like.

Suitable polypeptides also include: CHM (choroidermia (Rab escortprotein 1)), a polypeptide that, when defective or missing, causeschoroideremia (see, e.g., Donnelly et al. (1994) Hum. Mol. Genet.3:1017; and van Bokhoven et al. (1994) Hum. Mol. Genet. 3:1041); andCrumbs homolog 1 (CRB 1), a polypeptide that, when defective or missing,causes Leber congenital amaurosis and retinitis pigmentosa (see, e.g.,den Hollander et al. (1999) Nat. Genet. 23:217; and GenBank AccessionNo. CAM23328).

Suitable polypeptides also include polypeptides that, when defective ormissing, lead to achromotopsia, where such polypeptides include, e.g.,cone photoreceptor cGMP-gated channel subunit alpha (CNGA3) (see, e.g.,GenBank Accession No. NP_001289; and Booij et al. (2011) Ophthalmology118:160-167); cone photoreceptor cGMP-gated cation channel beta-subunit(CNGB3) (see, e.g., Kohl et al. (2005) Eur J Hum Genet. 13(3):302);guanine nucleotide binding protein (G protein), alpha transducingactivity polypeptide 2 (GNAT2) (ACHM4); and ACHM5; and polypeptidesthat, when defective or lacking, lead to various forms of colorblindness (e.g., L-opsin, M-opsin, and S-opsin). See Mancuso et al.(2009) Nature 461(7265):784-787.

Site-Specific Endonucleases

In some cases, a gene product of interest is a site-specificendonuclease that provide for site-specific knock-down of gene function,e.g., where the endonuclease knocks out an allele associated with aretinal disease. For example, where a dominant allele encodes adefective copy of a gene that, when wild-type, is a retinal structuralprotein and/or provides for normal retinal function, a site-specificendonuclease can be targeted to the defective allele and knock out thedefective allele.

In addition to knocking out a defective allele, a site-specific nucleasecan also be used to stimulate homologous recombination with a donor DNAthat encodes a functional copy of the protein encoded by the defectiveallele. Thus, e.g., a subject rAAV virion can be used to deliver both asite-specific endonuclease that knocks out a defective allele, and canbe used to deliver a functional copy of the defective allele, resultingin repair of the defective allele, thereby providing for production of afunctional retinal protein (e.g., functional retinoschisin, functionalRPE65, functional peripherin, etc.). See, e.g., Li et al. (2011) Nature475:217. In some embodiments, a subject rAAV virion comprises aheterologous nucleotide sequence that encodes a site-specificendonuclease; and a heterologous nucleotide sequence that encodes afunctional copy of a defective allele, where the functional copy encodesa functional retinal protein. Functional retinal proteins include, e.g.,retinoschisin, RPE65, retinitis pigmentosa GTPase regulator(RGPR)-interacting protein-1, peripherin, peripherin-2, and the like.

Site-specific endonucleases that are suitable for use include, e.g.,zinc finger nucleases (ZFNs); and transcription activator-like effectornucleases (TALENs), where such site-specific endonucleases arenon-naturally occurring and are modified to target a specific gene. Suchsite-specific nucleases can be engineered to cut specific locationswithin a genome, and non-homologous end joining can then repair thebreak while inserting or deleting several nucleotides. Suchsite-specific endonucleases (also referred to as “INDELs”) then throwthe protein out of frame and effectively knock out the gene. See, e.g.,U.S. Patent Publication No. 2011/0301073.

Regulatory Sequences

In some embodiments, a nucleotide sequence encoding a gene product ofinterest is operably linked to a constitutive promoter. In otherembodiments, a nucleotide sequence encoding a gene product of interestis operably linked to an inducible promoter. In some instances, anucleotide sequence encoding a gene product of interest is operablylinked to a tissue-specific or cell type-specific regulatory element.For example, in some instances, a nucleotide sequence encoding a geneproduct of interest is operably linked to a photoreceptor-specificregulatory element (e.g., a photoreceptor-specific promoter), e.g., aregulatory element that confers selective expression of the operablylinked gene in a photoreceptor cell. Suitable photoreceptor-specificregulatory elements include, e.g., a rhodopsin promoter; a rhodopsinkinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); abeta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med.9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007)supra); an interphotoreceptor retinoid-binding protein (IRBP) geneenhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyamaet al. (1992) Exp Eye Res. 55:225).

Pharmaceutical Compositimns

The present disclosure provides a pharmaceutical composition comprising:a) a subject rAAV virion, as described above; and b) a pharmaceuticallyacceptable carrier, diluent, excipient, or buffer. In some embodiments,the pharmaceutically acceptable carrier, diluent, excipient, or bufferis suitable for use in a human.

Such excipients, carriers, diluents, and buffers include anypharmaceutical agent that can be administered without undue toxicity.Pharmaceutically acceptable excipients include, but are not limited to,liquids such as water, saline, glycerol and ethanol. Pharmaceuticallyacceptable salts can be included therein, for example, mineral acidsalts such as hydrochlorides, hydrobromides, phosphates, sulfates, andthe like; and the salts of organic acids such as acetates, propionates,malonates, benzoates, and the like. Additionally, auxiliary substances,such as wetting or emulsifying agents, pH buffering substances, and thelike, may be present in such vehicles. A wide variety ofpharmaceutically acceptable excipients are known in the art and need notbe discussed in detail herein. Pharmaceutically acceptable excipientshave been amply described in a variety of publications, including, forexample, A. Gennaro (2000) “Remington: The Science and Practice ofPharmacy,” 20th edition, Lippincott, Williams, & Wilkins; PharmaceuticalDosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds.,7^(th) ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed.Amer. Pharmaceutical Assoc.

Methods of Delivering a Gene Product to a Retinal Cell and TreatmentMethods

The present disclosure provides a method of delivering a gene product toa retinal cell in an individual, the method comprising administering tothe individual a subject rAAV virion as described above. The geneproduct can be a polypeptide or an interfering RNA (e.g., an shRNA, ansiRNA, and the like), an aptamer, or a site-specific endonuclease, asdescribed above. Delivering a gene product to a retinal cell can providefor treatment of a retinal disease. The retinal cell can be aphotoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell,an amacrine cell, a horizontal cell, or a retinal pigmented epithelialcell. In some cases, the retinal cell is a photoreceptor cell, e.g., arod or cone cell.

The present disclosure provides a method of treating a retinal disease,the method comprising administering to an individual in need thereof aneffective amount of a subject rAAV virion as described above. A subjectrAAV virion can be administered via intraocular injection, byintravitreal injection, or by any other convenient mode or route ofadministration. Other convenient modes or routes of administrationinclude, e.g., intravenous, intranasal, etc.

A “therapeutically effective amount” will fall in a relatively broadrange that can be determined through experimentation and/or clinicaltrials. For example, for in vivo injection, i.e., injection directlyinto the eye, a therapeutically effective dose will be on the order offrom about 10⁶ to about 10¹⁵ of the rAAV virions, e.g., from about 10⁸to 10¹² rAAV virions. For in vitro transduction, an effective amount ofrAAV virions to be delivered to cells will be on the order of from about10⁸ to about 10¹³ of the rAAV virions. Other effective dosages can bereadily established by one of ordinary skill in the art through routinetrials establishing dose response curves.

In some embodiments, more than one administration (e.g., two, three,four or more administrations) may be employed to achieve the desiredlevel of gene expression over a period of various intervals, e.g.,daily, weekly, monthly, yearly, etc.

Ocular diseases that can be treated using a subject method include, butare not limited to, acute macular neuroretinopathy; Behcet's disease;choroidal neovascularization; diabetic uveitis; histoplasmosis; maculardegeneration, such as acute macular degeneration, non-exudative agerelated macular degeneration and exudative age related maculardegeneration; edema, such as macular edema, cystoid macular edema anddiabetic macular edema; multifocal choroiditis; ocular trauma whichaffects a posterior ocular site or location; ocular tumors; retinaldisorders, such as central retinal vein occlusion, diabetic retinopathy(including proliferative diabetic retinopathy), proliferativevitreoretinopathy (PVR), retinal arterial occlusive disease, retinaldetachment, uveitic retinal disease; sympathetic opthalmia; VogtKoyanagi-Harada (VKH) syndrome; uveal diffusion; a posterior ocularcondition caused by or influenced by an ocular laser treatment;posterior ocular conditions caused by or influenced by a photodynamictherapy; photocoagulation, radiation retinopathy; epiretinal membranedisorders; branch retinal vein occlusion; anterior ischemic opticneuropathy; non-retinopathy diabetic retinal dysfunction; retinoschisis;retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy;Stargardt disease (fundus flavimaculatus); inherited maculardegeneration; chorioretinal degeneration; Leber congenital amaurosis;congenital stationary night blindness; choroideremia; Bardet-Biedlsyndrome; macular telangiectasia; Leber's hereditary optic neuropathy;retinopathy of prematurity; and disorders of color vision, includingachromatopsia, protanopia, deuteranopia, and tritanopia.

Nucleic Acids and Host Cells

The present disclosure provides an isolated nucleic acid comprising anucleotide sequence that encodes a subject variant adeno-associatedvirus (AAV) capsid protein as described above, where the variant AAVcapsid protein comprises an insertion of from about 5 amino acids toabout 11 amino acids in the GH loop or loop IV relative to acorresponding parental AAV capsid protein, and where the variant capsidprotein, when present in an AAV virion, provides for increasedinfectivity of a retinal cell compared to the infectivity of the retinalcell by an AAV virion comprising the corresponding parental AAV capsidprotein. A subject isolated nucleic acid can be an AAV vector, e.g., arecombinant AAV vector.

Insertion Peptides

A variant AAV capsid protein encoded by a subject nucleic acid has aninsertion peptide of from about 5 amino acids to about 11 amino acids inlength is inserted into the GH loop of an AAV capsid. The insertionpeptide has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8amino acids, 9 amino acids, 10 amino acids, or 11 amino acids.

The insertion peptide can comprise an amino acid sequence of any one ofthe formulas set forth below.

For example, an insertion peptide can be a peptide of from 5 to 11 aminoacids in length, where the insertion peptide is of Formula I:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is selected from Leu, Asn, and Lys;

X₂ is selected from Gly, Glu, Ala, and Asp;

X₃ is selected from Glu, Thr, Gly, and Pro;

X₄ is selected from Thr, Ile, Gln, and Lys;

X₅ is selected from Thr and Ala;

X₆ is selected from Arg, Asn, and Thr;

X₇, if present, is selected from Pro and Asn.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula Ha:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

each of X₁-X₄ is any amino acid;

X₅ is Thr;

X₆ is Arg; and

X₇ is Pro.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula IIb:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is selected from Leu and Asn;

X₂, if present, is selected from Gly and Glu;

X₃ is selected from Glu and Thr;

X₄ is selected from Thr and Ile;

X₅ is Thr;

X₆ is Arg; and

X₇ is Pro.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula II:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is Lys;

X₂ is selected from Ala and Asp;

X₃ is selected from Gly and Pro;

X₄ is selected from Gln and Lys;

X₅ is selected from Thr and Ala;

X₆ is selected from Asn and Thr;

X₇, if present, is Asn.

As another example, an insertion peptide can be a peptide of from 5 to11 amino acids in length, where the insertion peptide is of Formula IV:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ Y ₄

where:

each of Y₁-Y₄, if present, is independently selected from Ala, Leu, Gly,Ser, and Thr;

X₁, if present, is a positively charged amino acid or an uncharged aminoacid; or is selected from Leu, Asn, Arg, Ala, Ser, and Lys;

X₂ is a negatively charged amino acid or an uncharged amino acid; or isselected from Gly, Glu, Ala, Val, Thr, and Asp;

X₃ is a negatively charged amino acid or an uncharged amino acid; or isselected from Glu, Thr, Gly, Asp, or Pro;

X₄ is selected from Thr, Ile, Gly, Lys, Asp, and Gln;

X₅ is a polar amino acid, an alcohol (an amino acid having a freehydroxyl group), or a hydrophobic amino acid; or is selected from Thr,Ser, Val, and Ala;

X₆ is a positively charged amino acid or an uncharged amino acid; or isselected from Arg, Val, Lys, Pro, Thr, and Asn; and

X₇, if present, is a positively charged amino acid or an uncharged aminoacid; or is selected from Pro, Gly, Phe, Asn, and Arg.

As non-limiting examples, the insertion peptide can comprise an aminoacid sequence selected from LGETIRP (SEQ ID NO:13), NETITRP (SEQ IDNO:14), KAGQANN (SEQ ID NO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ IDNO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQID NO:60).

In some cases, the insertion peptide has from 1 to 4 spacer amino acids(Y₁-Y₄) at the amino terminus and/or at the carboxyl terminus of any oneof LGETTRP (SEQ ID NO:13), NETITRP (SEQ ID NO:14), KAGQANN (SEQ IDNO:15), KDPKTTN (SEQ ID NO:16), KDTDTTR (SEQ ID NO:57), RAGGSVG (SEQ IDNO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60). Suitablespacer amino acids include, but are not limited to, leucine, alanine,glycine, and serine.

For example, in some cases, an insertion peptide has one of thefollowing amino acid sequences: LALGETTRPA (SEQ ID NO:45); LANETITRPA(SEQ ID NO:46), LAKAGQANNA (SEQ ID NO:47), LAKDPKTTNA (SEQ ID NO:48),LAKDTDTTRA (SEQ ID NO:61), LARAGGSVGA (SEQ ID NO:62), LAAVDTTKFA (SEQ IDNO:63), and LASTGKVPNA (SEQ ID NO:64). As another example, in somecases, an insertion peptide has one of the following amino acidsequences: AALGETTRPA (SEQ ID NO:49); AANETITRPA (SEQ ID NO:50),AAKAGQANNA (SEQ ID NO:51), and AAKDPKTTNA (SEQ ID NO:52). As yet anotherexample, in some cases, an insertion peptide has one of the followingamino acid sequences: GLGETTRPA (SEQ ID NO:53); GNETITRPA (SEQ IDNO:54), GKAGQANNA (SEQ ID NO:55), and GKDPKTTNA (SEQ ID NO:56). Asanother example, in some cases, an insertion peptide comprises one ofKDTDTR (SEQ ID NO:57), RAGGSVG (SEQ ID NO:58), AVDTTKF (SEQ ID NO:59),and STGKVPN (SEQ ID NO:60), flanked on the C-terminus by AA and on theN-terminus by A; or comprises one of KDTDTTR (SEQ ID NO:57), RAGGSVG(SEQ ID NO:58), AVDTTKF (SEQ ID NO:59), and STGKVPN (SEQ ID NO:60)flanked on the C-terminus by G and on the N-terminus by A.

A subject recombinant AAV vector can be used to generate a subjectrecombinant AAV virion, as described above. Thus, the present disclosureprovides a recombinant AAV vector that, when introduced into a suitablecell, can provide for production of a subject recombinant AAV virion.

The present invention further provides host cells, e.g., isolated(genetically modified) host cells, comprising a subject nucleic acid. Asubject host cell can be an isolated cell, e.g., a cell in in vitroculture. A subject host cell is useful for producing a subject rAAVvirion, as described below. Where a subject host cell is used to producea subject rAAV virion, it is referred to as a “packaging cell.” In someembodiments, a subject host cell is stably genetically modified with asubject nucleic acid. In other embodiments, a subject host cell istransiently genetically modified with a subject nucleic acid.

A subject nucleic acid is introduced stably or transiently into a hostcell, using established techniques, including, but not limited to,electroporation, calcium phosphate precipitation, liposome-mediatedtransfection, and the like. For stable transformation, a subject nucleicacid will generally further include a selectable marker, e.g., any ofseveral well-known selectable markers such as neomycin resistance, andthe like.

A subject host cell is generated by introducing a subject nucleic acidinto any of a variety of cells, e.g., mammalian cells, including, e.g.,murine cells, and primate cells (e.g., human cells). Suitable mammaliancells include, but are not limited to, primary cells and cell lines,where suitable cell lines include, but are not limited to, 293 cells,COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2fibroblasts, CHO cells, and the like. Non-limiting examples of suitablehost cells include, e.g., HeLa cells (e.g., American Type CultureCollection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61,CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells(e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No.CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No.CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonickidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Asubject host cell can also be made using a baculovirus to infect insectcells such as Sf9 cells, which produce AAV (see, e.g., U.S. Pat. No.7,271,002; U.S. patent application Ser. No. 12/297,958)

In some embodiments, a subject genetically modified host cell includes,in addition to a nucleic acid comprising a nucleotide sequence encodinga variant AAV capsid protein, as described above, a nucleic acid thatcomprises a nucleotide sequence encoding one or more AAV rep proteins.In other embodiments, a subject host cell further comprises an rAAVvector. An rAAV virion can be generated using a subject host cell.Methods of generating an rAAV virion are described in, e.g., U.S. PatentPublication No. 2005/0053922 and U.S. Patent Publication No.2009/0202490.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 AAV Variant with Enhanced Transduction of Retinal Cells

The approach used was to create a peptide display library by introducinga unique AvrII site into the wild type AAV2 genome between amino acid587 and 588 by polymerase chain reaction (PCR) mutagenesis. A random 21nucleotide insert, 7mer For, was used to synthesize dsDNA inserts, alongwith the antisense primer 7mer Rev. The resulting dsDNA inserts werecloned into the AvrII site of the genome after digestion with NheI,producing a diverse 7mer display library which was then packaged (Peraboet al., 2003; Muller et al., 2003). The virus was generated such thateach viral genome was packaged or encapsidated within the capsid proteinvariant that that genome encoded. In this respect, functionalimprovements identified through selection can be linked to the genomesequence encoding this improved function contained within the viralcapsid.

This library was subjected to positive selection within rho-GFP mice(Wensel et al. (2005) Vision Res. 45:3445). Briefly, in one round ofselection, adult rho-GFP mice were intravitreally injected with 2 μL ofphosphate buffered saline (PBS)-dialyzed, iodixanol-purified librarywith a genomic titer of approximately 1×10¹² viral genomes (vg)/mL. Anultrafine 30½-gauge disposable needle was passed through the sclera ofthe animal's eye, at the equator and next to the limbus, into thevitreous cavity. Injection of 2 μl of virus was made with directobservation of the needle in the center of the vitreous cavity. One weekpost-injection, eyes were enucleated and retinas dissociated using alight, papain protease treatment, followed by fluorescence activatedcell sorter (FACS) isolation of photoreceptor populations. Successfulvirions were then PCR amplified from subsequent genomic extractions andfurther cloned and repackaged for injection.

Further iterations of this selection were performed, narrowing the poolof variants to a subset with the most permissive mutations. After threeiterations, a round of error-prone PCR was performed to create a furthergeneration of variants for selection. In total, this process wasrepeated for two generations. In this respect, this process of directedevolution created photoreceptor-permissive AAV variants through theapplication of positive selection and induced mutagenesis, similar tothe process of natural evolution.

Subsequently, the cap genes of fifty variants were sequenced todetermine the most prominent and successful variants to have permissivemutations for intravitreal photoreceptor transduction. Of the 50 clones,46 gave readable sequences of a 7mer insert. Remarkably, nearly twothirds of clones contained the same distinct 7mer motif (˜⁵⁸⁸LGETTRP˜;SEQ ID NO:13). Interestingly, the next most prominent variant(˜⁵⁸⁸NETITRP˜; SEQ ID NO: 14) also contained a similar flanking motifconsisting of a positively-charged arginine residue in between a polarthreonine and a nonpolar proline residue (TRP).

TABLE 1  Appr. Frequency Clone (%) Frequency ~⁵⁸⁸LGETTRP~(SEQ ID NO: 13) 64 31 ~⁵⁸⁸NETITRP~ (SEQ ID NO: 14) 12 5 ~⁵⁸⁸KAGQANN~(SEQ ID NO: 15) 6 3 ~⁵⁸⁸KDPKTTN~ (SEQ ID NO: 16) 4 2~⁵⁸⁸KDTDTTR (SEQ ID NO: 57) 2 ~⁵⁸⁸RAGGSVG (SEQ ID NO: 58) 1~⁵⁸⁸AVDTTKF (SEQ ID NO: 59) 1 ~⁵⁸⁸STGKVPN (SEQ ID NO: 60) 1

Table 1 Sequencing of isolated variants from directed evolution revealsa high degree of convergence in viral libraries. All variants derivedfrom the AAV2 7mer library, with approximately 64% of variantscontaining the same 7mer motif (˜⁵⁸⁸LGETTRP˜ (SEQ ID NO:13)).

Among the 7mer insert sequences, there were moderate preferences atparticular positions, e.g., a positively charged amino acid at position1; a negatively charged amino acid at position 2; an alcohol (e.g., anamino acid having an alcohol group (a free hydroxyl group), such as Thror Ser) at position 5.

The 7mer inserts were flanked by spacers, as shown in Table 2:

Clone Frequency ~⁵⁸⁸LALGETTRPA~ (SEQ ID NO: 45) 31 ~⁵⁸⁸LANETITRPA~(SEQ ID NO: 46) 5 ~⁵⁸⁸LAKAGQANNA~ (SEQ ID NO: 47) 3 ~⁵⁸⁸LAKDPKTTNA~(SEQ ID NO: 48) 2 ~⁵⁸⁸LAKDTDTTRA~ (SEQ ID NO: 61) 2 ~⁵⁸⁸LARAGGSVGA~(SEQ ID NO: 62) 1 ~⁵⁸⁸LAAVDTTKFA~ (SEQ ID NO: 63) 1 ~⁵⁸⁸LASTGKVPNA~(SEQ ID NO: 64) 1

FIG. 1. Representative three-dimensional capsid model of AAV2 containinga random heptamer (shown in orange) following amino acid 587. This areaof the AAV2 capsid likely participates in cell-surface receptor binding.

In light of the high degree of library convergence from theabove-described selection, a recombinant form of AAV2 ˜⁵⁸⁸LGETTRP˜ (SEQID NO:13; nick named 7M8) was cloned and packaged the vector with ascCAG-GFP transgene to visualize its transduction profile. Three weeksfollowing intravitreal injection in adult mice, robust expression innumerous cell types, including retinal ganglion cells (RGCs) and Millercells, was observed. Importantly, transduction of photoreceptors inretinas injected with 7M8, as seen by GFP expression in outer nuclearlayer (ONL) nuclei (red arrows) and in outer segments (FIG. 2, bluearrow), was observed, whereas AAV2 showed no discernable photoreceptorexpression.

FIG. 2 AAV2 7M8 variant (right) demonstrates greater levels ofintravitreal photoreceptor transduction relative to AAV2 (left).Confocal microscopy of transverse retinal sections three weeks afterintravitreal injection of 2 μL of 1×10¹² vg/mL of AAV2 7M8 and AAV2scCAG GFP in adult mice. Red arrows (top) denote photoreceptor nucleiand blue arrow (top) denote photoreceptor outer segments.

In light of these gains in retinal cell transduction, an attempt wasmade to increase specificity in expression through the use of assRho-eGFP transgene containing a photoreceptor-specific rhodopsinpromoter to better resolve transduction efficiencies specifically inphotoreceptors (FIG. 3). Indeed the use of a photoreceptor specific Rhopromoter limited the GFP expression to the photoreceptors. An attemptwas made to improve 7M8 transduction efficiency by combining a rationaldesign approach to the previous directed evolution approach. Therefore,four surface exposed tyrosine residues were mutagenized tophenylalanines on the 7M8 capsid (Y273F, Y444F, Y500F, and Y730F) whichhas previous been shown to increase photoreceptor infectivity(Petrs-Silva et al., 2009). Interestingly, the addition of mutationsdecreased number of photoreceptors transduced compared to the originalvirus as show by FACs sorting of the GFP(+) photoreceptors from 7m8 or7m8-4YF infected retinas (FIG. 4).

FIG. 3. Representative fluorescence images of retinal cryoslices showingGFP expression resulting from 7m8 carrying the GFP gene under thecontrol of the ubiquitous CAG promoter (left) or a photoreceptorspecific Rho promoter (right).

FIG. 4. GFP(+) photoreceptor cells per million retinal cells as countedby flow cytometry. 7m8 transduces more than 2× the amount ofphotoreceptors compared 7m8 bearing 4 tyrosine mutations (top).

Example 2 Treatment of Retinoschisis

Using the expression construct 7m8-rho-RS1, a functional retinoschisin(RS1) protein was delivered to retinoschisin-deficient mice(Rs1h-deficient mice; Rs1h is the mouse homolog of human RS1). Thevector comprises a nucleotide sequence encoding a functionalretinoschisin protein under transcriptional control of a rhodopsinpromoter. See FIGS. 13A-C, where the bold and underlined nucleotidesequence (nucleotides 4013-4851) are the rhodopsin promoter, andnucleotides 4866-5540 (with the start atg and stop tga sequences shownin bold) encode a human RS1 protein.

The 7m8-rho-RS1 construct was administered intravitreally to Rs1h−/−mice at P15. Rs1h−/− mice were generated through targeted disruption ofexon 3 of the Rs1h gene, as described (Weber et al. (2002) Proc. Natl.Acad. Sci. USA 99:6222). The Rs1h−/− mice are deficient in the Rs1hprotein product, have an electronegative ERG (e.g., a reduced b-wavewith relative preservation of the a-wave) and splitting of the layers ofthe retina, similar to what is seen in human retinoschisis patients.Injection of the 7m8-rho-RS1 vector into the Rs1h−/− led to high levelsof panretinal RS1 expression from photoreceptors in the retina. RS1expression led to improved retinal morphology with a decrease in thenumber and size of cavities in the retina as seen in spectral-domainoptical coherence tomography (SD-OCT) imaging (FIGS. 7A-I), a rescue ofthe ERG b-wave (FIGS. 8A-D), and long-term structural preservation ofthe retina (FIGS. 9A-E).

FIGS. 7A-I. Representative high-resolution SD-OCT images of retinasinjected with 7m8-rho-GFP (left column), 7m8-rho-RS1 (middle column), oruninjected WT animals (right column). Fundus images were taken throughthe inner nuclear layer of the superior retina and exclude other layers(a-c). Transverse images of the superior (d-f) and inferior (g-i) retinawere taken using the optic nerve head as a landmark.

The untreated RS1 retina increases in overall thickness when measuredfrom the inner limiting membrane (ILM) to the photoreceptors, as thepathology progresses due to the schisis splitting the inner retina. Thisprocess is distinct from that observed in most retinal degenerativediseases (RDD) which do not form schisis, but exhibit progressivephotoreceptor cell death in the INL and concomitant retinal thinning andloss of ERG amplitude. In RS1, the ONL thins as photoreceptors die fromthe disease, but this is distinct from the overall retinal thicknesschange. It is generally thought that a successful therapy for RS1 wouldreturn the overall retinal thickness to the wildtype and ameliorate thephotoreceptor loss in the ONL. In most RDD other than Rs1, the loss ofphotoreceptors, marked by ONL thinning, is paralleled by a decrease inretinal physiological output as measured by the ERG amplitude. RS1 isone of the very few examples of a retinal disease in which the pathologyincreases the retinal thickness with concomitant erg amplitude loss. Insummary, restoring the RS1 gene product, an extracellular retinal“glue;—thins the retina back to the wildtype thickness and the ergamplitude returns to near normal levels as the schisis resolves.

FIG. 8a shows a comparison of functional rescue of untreated Rs1−/− eyesto AAV2-rho-RS1, 7m8-rho-GFP, and 7m8-rho-RS1 injected eyes both onemonth (left) and 4 months (right) after injection. One monthpost-injection, 7m8-rho-RS1 led to considerable rescue of the ERG b-waveamplitude, whereas AAV2-rho.RS1 was statistically indistinguishable fromuntreated eyes.

After 4 months, the 7m8-rho-RS1 amplitude further increases toward thewild-type amplitude (right). FIG. 8b shows representative ERG tracesfrom 7m8-rho-RS1-injected eyes show improved amplitude of the a-wave andb-wave and a waveform closer to wild-type eyes, compared to7m8-rho-GFP-injected eyes. FIG. 8c shows the amplitude of the full-fieldscotopic b-wave resulting from a high intensity (1 log cd×s/m2) stimuluswas recorded on a monthly basis beginning one month after injection atP15 for each condition. Three responses were recorded and averaged foreach eye at each time point.

Mean ERG b-wave amplitudes were plotted as a function of timepost-injection. n=7 was used for both conditions. FIG. 8d shows ananalysis of ERG responses under scotopic (upper traces, stimulus rangefrom −3 to 1 log cd×s/m2) and photopic (lower traces, range from −0.9 to1.4 log cd×s/m2) conditions indicates improved rod and cone functionover a range of stimuli intensities.

FIGS. 9A-E. Sustained improvements in retinal thickness measured at 10months post 7m8-rho-RS1 treatment. Representative transverse SD-OCTimages of a) 7m8-rho-RS1 or b) or 7m8-rho-GFP treated retinas 10 monthspost-injection centered on the optic nerve head. Measurements of c)retinal thickness, d) ONL thickness, and e) and inner and outer segmentthickness are plotted as a function of distance from the optic nervehead.

Example 3 AAV Variant Used to Deliver a Protein to Retinal Cells in theMacaque

A recombinant AAV2 virion (7m8 carrying GFP under the control of aconnexin36 promoter) was generated. The recombinant AAV2 virion includedan AAV2 capsid variant with an insertion of LALGETTRPA peptide betweenamino acids 587 and 588 of AAV2 capsid, and GFP under transcriptionalcontrol of a connexin36 promoter, which is expressed in interneurons.The rAAV2 virion was injected intravitreally into the eye of a macaque.The data are shown in FIG. 18.

FIG. 18 provides a fluorescence fundus image showing GFP expression atthe back of the retina 9 weeks after administration of 7m8 carrying GFPunder the control of a connexin36 promoter. Compared to the parentalAAV2 serotype (Yin et al, IOVS 52(5); 2775), a higher level ofexpression was seen in the foveal ring, and visible fluorescence wasseen in the central retina outside the fovea.

REFERENCES

-   Daiger S P, Bowne S J, Sullivan L S (2007) Perspective on genes and    mutations causing retinitis pigmentosa. Arch Ophthalmol 125:    151-158.-   Dalkara D, Kolstad K D, Caporale N, Visel M, Klimczak R R, et    al. (2009) Inner Limiting Membrane Barriers to AAV Mediated Retinal    Transduction from the Vitreous. Mol Ther.-   den Hollander A I, Roepman R, Koenekoop R K, Cremers F P (2008)    Leber congenital amaurosis: genes, proteins and disease mechanisms.    Prog Retin Eye Res 27: 391-419.-   Gruter O, Kostic C, Crippa S V, Perez M T, Zografos L, et al. (2005)    Lentiviral vector-mediated gene transfer in adult mouse    photoreceptors is impaired by the presence of a physical barrier.    Gene Ther 12: 942-947.-   Maguire A M, Simonelli F, Pierce E A, Pugh E N, Jr., Mingozzi F, et    al. (2008) Safety and efficacy of gene transfer for Leber's    congenital amaurosis. N Engl J Med 358: 2240-2248.-   Mancuso K, Hauswirth W W, Li Q, Connor T B, Kuchenbecker J A, et    al. (2009) Gene therapy for red-green colour blindness in adult    primates. Nature 461: 784-787.-   McGee Sanftner L H, Abel H, Hauswirth W W, Flannery J G (2001) Glial    cell line derived neurotrophic factor delays photoreceptor    degeneration in a transgenic rat model of retinitis pigmentosa. Mol    Ther 4: 622-629.-   Muller O J, Kaul F, Weitzman M D, Pasqualini R, Arap W, et    al. (2003) Random peptide libraries displayed on adeno-associated    virus to select for targeted gene therapy vectors. Nat Biotechnol    21: 1040-1046.-   Nakazawa T. et al. (2007) Attenuated glial reactions and    photoreceptor degeneration after retinal detachment in mice    deficient in glial fibrillary acidic protein and vimentin. Invest    Ophthamol Vis Sci 48: 2760-8.-   Nakazawa T. et al. (2006) Characterization of cytokine responses to    retinal detachment in rats. Mol Vis 12: 867-78.-   Perabo L, Buning H, Kofler D M, Ried M U, Girod A, et al. (2003) In    vitro selection of viral vectors with modified tropism: the    adeno-associated virus display. Mol Ther 8: 151-157.-   Petrs-Silva H, Dinculescu A, Li Q, Min S H, Chiodo V, et al. (2009)    High-efficiency transduction of the mouse retina by tyrosine-mutant    AAV serotype vectors. Mol Ther 17: 463-471.-   Reme C E, Grimm C, Hafezi F, Wenzel A, Williams T P (2000) Apoptosis    in the Retina: The Silent Death of Vision. News Physiol Sci 15:    120-124.-   Rolling F (2004) Recombinant AAV-mediated gene transfer to the    retina: gene therapy perspectives. Gene Ther 11 Suppl 1: S26-32.-   Wensel T G, Gross A K, Chan F, Sykoudis K, Wilson J H (2005)    Rhodopsin-EGFP knock-ins for imaging quantal gene alterations.    Vision Res 45: 3445-3453.-   Zhong L, Li B, Mah C S, Govindasamy L, Agbandje-McKenna M, et    al. (2008) Next generation of adeno-associated virus 2 vectors:    point mutations in tyrosines lead to high-efficiency transduction at    lower doses. Proc Natl Acad Sci USA 105: 7827-7832.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A recombinant adeno-associated virus (rAAV) virion comprising: a) avariant AAV capsid protein, wherein the variant AAV capsid proteincomprises a peptide insertion relative to a corresponding parental AAVcapsid protein, wherein the peptide insertion comprises the amino acidsequence LGETTRP (SEQ ID NO: 13), wherein the insertion site is locatedbetween two adjacent amino acids at a position between amino acids 570and 611 of VP1 of AAV2 or the corresponding position in the capsidprotein of another AAV serotype, and wherein the variant capsid proteinconfers increased infectivity of a retinal cell compared to theinfectivity of the retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein; and b) a heterologous nucleicacid comprising a nucleotide sequence encoding retinoschisin. 2.(canceled)
 3. (canceled)
 4. The rAAV virion of claim 1, wherein theretinal cell is a photoreceptor, a retinal ganglion cell, a Müller cell,a bipolar cell, an amacrine cell, a horizontal cell, or a retinalpigmented epithelium cell.
 5. The rAAV virion of claim 1, wherein theinsertion site is located between amino acids corresponding to aminoacids 587 and 588 of VP1 of AAV2 or the corresponding position in thecapsid protein of another AAV serotype.
 6. The rAAV virion of claim 1,wherein the rAAV virion exhibits at least 10-fold increased infectivityof a retinal cell compared to the infectivity of the retinal cell by anAAV virion comprising the corresponding parental AAV capsid protein. 7.The rAAV virion of claim 1, wherein the rAAV virion exhibits at least50-fold increased infectivity of a retinal cell compared to theinfectivity of the retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein.
 8. (canceled)
 9. (canceled)10. (canceled)
 11. A pharmaceutical composition comprising: a) arecombinant adeno-associated virus virion of claim 1; and b) apharmaceutically acceptable excipient.
 12. A method of deliveringretinoschisin to a retinal cell in an individual, the method comprisingadministering to the individual a recombinant adeno-associated virus(rAAV) virion according to claim
 1. 13. (canceled)
 14. (canceled) 15.(canceled)
 16. (canceled)
 17. A method of treating retinoschisis, themethod comprising administering to an individual in need thereof aneffective amount of a recombinant adeno-associated virus (rAAV) virionaccording to claim
 1. 18. The method of claim 17, wherein saidadministering is by intraocular injection.
 19. The method of claim 17,wherein said administering is by intravitreal injection.
 20. (canceled)21. An isolated nucleic acid comprising a nucleotide sequence thatencodes a variant adeno-associated virus (AAV) capsid protein, whereinthe variant AAV capsid protein comprises a peptide insertion of relativeto a corresponding parental AAV capsid protein, and wherein the peptideinsertion comprises the amino acid sequence LGETTRP (SEQ ID NO: 13),wherein the insertion site is located between two adjacent amino acidsat a position between amino acids 570 and 611 of VP1 of AAV2 or thecorresponding position in the capsid protein of another AAV serotype,and wherein the variant capsid protein confers increased infectivity ofa retinal cell compared to the infectivity of the retinal cell by an AAVvirion comprising the corresponding parental AAV capsid protein.
 22. Theisolated nucleic acid of claim 21, wherein the insertion site is locatedbetween amino acids corresponding to amino acids 587 and 588 of VP ofAAV2 or the corresponding position in the capsid protein of another AAVserotype.
 23. An isolated, genetically modified host cell comprising thenucleic acid of claim
 21. 24. A variant adeno-associated virus (AAV)capsid protein, wherein the variant AAV capsid protein comprises apeptide insertion of relative to a corresponding parental AAV capsidprotein, and wherein the peptide insertion comprises the amino acidsequence LGETTRP (SEQ ID NO: 13), wherein the insertion site is locatedbetween two adjacent amino acids at a position between amino acids 570and 611 of VP1 of AAV2 or the corresponding position in the capsidprotein of another AAV serotype, and wherein the variant capsid proteinconfers increased infectivity of a retinal cell compared to theinfectivity of the retinal cell by an AAV virion comprising thecorresponding parental AAV capsid protein.
 25. (canceled)
 26. Thevariant AAV capsid protein of claim 24, wherein the insertion site is orthe corresponding position in the capsid protein of another AAVserotype.
 27. A nucleic acid comprising a nucleotide sequence encodingthe variant AAV capsid protein of claim
 24. 28. The rAAV virion of claim1, wherein the retinoschisin encoded by the heterologous nucleic acidcomprises an amino acid sequence having at least 90% amino acid sequenceidentity to a contiguous stretch of from about 200 amino acids to 224amino acids of SEQ ID NO:
 10. 29. The rAAV virion of claim 1, whereinthe retinoschisin encoded by the heterologous nucleic acid is undertranscriptional control of a heterologous promoter.
 30. Thepharmaceutical composition of claim 11, wherein the retinoschisinencoded by the heterologous nucleic acid comprises an amino acidsequence having at least 90% amino acid sequence identity to acontiguous stretch of from about 200 amino acids to 224 amino acids ofSEQ ID NO:
 10. 31. The pharmaceutical composition of claim 11, whereinthe retinoschisin encoded by the heterologous nucleic acid is undertranscriptional control of a heterologous promoter.
 32. The method ofclaim 12, wherein the retinoschisin encoded by the heterologous nucleicacid comprises an amino acid sequence having at least 90% amino acidsequence identity to a contiguous stretch of from about 200 amino acidsto 224 amino acids of SEQ ID NO:
 10. 33. The method of claim 12, whereinthe retinoschisin encoded by the heterologous nucleic acid is undertranscriptional control of a heterologous promoter.
 34. The method ofclaim 17, wherein the retinoschisin encoded by the heterologous nucleicacid comprises an amino acid sequence having at least 90% amino acidsequence identity to a contiguous stretch of from about 200 amino acidsto 224 amino acids of SEQ ID NO:
 10. 35. The method of claim 17, whereinthe retinoschisin encoded by the heterologous nucleic acid is undertranscriptional control of a heterologous promoter.